Biomarkers for diseases of the nervous system can benefit from advanced proteomic analysis of cerebral spinal fluid (CSF) but some basic biological and pre-analytical parameters of CSF collection and handling, and reproducibility and accuracy of proteomic bioanalysis must first be systematically evaluated. Label-free differential expression profiling involves separation of tryptic digests of the CSF proteome depleted of the most abundant proteins by cation exchange chromatography followed by liquid chromatography coupled online with mass spectrometry. The effect of time from lumbar puncture to freezing at different temperatures with variable blood contamination, freeze thaw cycles, circadian time of collection and other variables will be investigated. The project will generate best practices for future collections and develop a CSF Integrity test for stored samples. Best practices will be applied to biomarker discovery in three pilot case-control studies-frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and schizophrenia, to find new biomarkers and inform power calculations and platforms for future studies. A resource for the neuroscience community will be established with an annotated in-depth catalog of the CSF proteome and research tools to access information about CSF proteins. Finally, the catalog will include information on tryptic peptide for each proteins that will enable academic and pharma investigators to construct a panel of peptides for multiplexed measurements of CSF proteins of choice without the need for antibody by multiple reaction monitoring (MRM), and thereby promote and facilitate biomarker discovery in many diseases of the nervous system. PUBLIC HEALTH RELEVANCE: Biomarkers for diseases of the nervous system can benefit from advanced proteomic analysis of cerebral spinal fluid (CSF) but some basic biological and analytical parameters of CSF collection, handling, and analysis must first be systematically evaluated. The project will generate best practices for future collections and develop a CSF Integrity test for stored samples. Elaboration of an annotated CSF proteome and biomarker discovery in frontotemporal lobar degeneration (FTLD), Alzheimer's disease, and schizophrenia will accelerate biomarker discovery to speed therapeutic development for these major diseases.